Optically based biomolecular assay techniques such as optical microtiter plate reading and optical molecular imaging are very powerful tools for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vitro and in vivo. These techniques have been increasingly used to probe protein function and gene expression. Optically based techniques exhibit the great advantages of picosecond temporal resolution as important in functional imaging, submicron spatial resolution as important for in vivo microscopy, single molecule sensitivity, and minimal invasion. These techniques also offer the potential for simultaneous use of multiple and distinguishable probes as important in molecular imaging. They also offer safety in that ionizing radiation is obviated. These techniques have advanced over the past decade due to rapid developments in laser technology, sophisticated reconstruction algorithms and imaging software originally developed for non-optical, tomographic imaging modes such as CT and MRI.
Of the various optical imaging techniques investigated to date, near-infrared fluorescence (NIRF) imaging is of particular interest for non-invasive in vivo imaging because of the relatively low tissue absorbance, minimal autofluorescence of near-infrared (NIR) light, and deep tissue penetration of up to 6-8 centimeters. In near-infrared fluorescence imaging, a laser or appropriately filtered light is used as a source of fluorescence excitation. The excitation light travels through body tissues. When it encounters a near-infrared fluorescent molecule (“contrast agent” or “probe”), the excitation light is absorbed. The fluorescent molecule then emits light as fluorescence with a longer wavelength and therefore spectrally distinguishable from the excitation light. Despite good penetration of biological tissues by near-infrared light, conventional near-infrared fluorescence probes are subject to many of the same limitations encountered with other contrast agents, including low signal-to-noise ratios.
A number of NIRF contrast-enhanced optical imaging probes have been developed and evaluated in small animals. These studies have established the use of near-infrared optical imaging in diagnosis, molecular characterization, and monitoring of treatment response in a number of disease models. Nanoparticles have been increasingly used in a wide range of biomedical applications such as drug carriers and imaging agents. They are engineered materials with dimensions typically smaller than 100 nm, small enough to reach almost anywhere in the body and can be easily derivatized with a variety of targeting ligands, multiple imaging moieties for multiple modalities imaging, or loaded with multiple molecules of a contrast agent, providing a significant boost in signal intensity for diverse imaging modalities. NIRF imaging based on nanoparticulate imaging probes is rapidly emerging as an advanced technology for noninvasive cancer detection, diagnostic and therapeutic applications. Nanoparticle-based imaging probes offer potential advantages over small molecule or low molecular weight polymer-based probes such as long circulating time for effective tumor delivery because small probes are subjected to fast excretion in vivo, given internal clearance of small molecules and reticuloendothelial system clearance of non-immunologically shielded compounds. Several reports have featured quantum dots (QDs) (Warren, C. W. et al. Science 1998, 281, 2016-2018) composed of a semiconductor core encapsulated within novel polymeric or lipid-based layers for NIRF optical imaging in cancer imaging in animals. However, most QDs are made of toxic material such as cadmium, and it has not yet been established that QDs are sufficiently stable to avoid becoming toxic in the body. The design and synthesis of smart nanoprobes is an enabler for NIRF imaging to be successful.
The principle of fluorescence resonance energy transfer (FRET) detection is based on the transfer of energy from excited donor dye molecules to acceptor dye molecules that are located in spatial proximity. FRET can be used to determine distance at a molecular level in a range between approximately 1 to 8 nm because the efficiency E of the energy transfer is very sensitive to the distance R between the donor and acceptor and declines proportionally to R06/(R06+R6), where R0 is the material-specific Förster radius defined as the distance at which the efficiency is 50%, and typically lies in the range of a few nanometers (less than 10 nm). Depending on the fluorescence quantum efficiency of the acceptor molecules, the energy transferred from the donor molecules to the acceptor molecules can either undergo nonradiative relaxation by means of internal conversion thereby leading to quenching of the donor energy, or can be emitted by means of fluorescence of the acceptor molecules. In the following portions of this specification, (a) the pairs of different molecules capable of acting as donors and acceptors for FRET are termed “FRET dye pairs”, and (b) the pairs of different nanoparticles comprised of one nanoparticle including FRET-capable donor molecules and a second, different nanoparticle including different, FRET-capable acceptor molecules, the pairs of different nanoparticles are termed “FRET-particle pairs”.
In biological systems, FRET is used to detect the mutual spatial proximity of appropriately labeled biomolecules. FRET can be used as a method for detecting protein-protein interactions, e.g., as a method for detecting an antigen-antibody reaction, a receptor-ligand interaction, a nucleic acid hybridization, hormone-receptor interaction or the binding of proteins to nucleic acids. The detection is itself effected by means of measuring the change in the intensity of, or the spectral change in, the donor fluorescence or acceptor fluorescence, or by means of measuring a change in the decay time of the donor fluorescence. A large number of applications in this regard are described in the literature, such as the detection of specific antigens in immunofluorescence assays (U.S. Pat. Nos. 3,996,345; 4,160,016; 4,174,384; 4,199,559).
Organic dye molecules that are used as labels and attached to biomolecules such as fluorescein, water soluble cyanine, or rhodamine, for example, are classical commercially available materials for making FRET dye pairs. A general disadvantage of these organic fluorescent dyes is that they frequently exhibit photostability that is inadequate for many applications. Particularly in the presence of oxygen or free radicals some of these dyes can be irreversibly damaged or destroyed after only a few million light absorption/light emission cycles. Also, some fluorescent dyes can have toxic effects on the biological materials in their vicinity. Furthermore, the fluorescent dyes used as labels often have very short blood circulation times making them inadequate for studying biological interactions that occur over time.
U.S. Pat. Nos. 5,326,692; 6,238,931; and 6,251,687 describe methods of using nanoparticles with FRET dye pairs in the same nanoparticle. The purpose of these methods is to provide a particle with a large net difference between the excitation wavelength and the emission wavelength (i.e., large net Stokes shift) to improve the signal-to-background figure of merit for fluorescent measurements, wherein the background is due to autofluorescence that typically has a relatively small Stokes shift. However the FRET is constant within the same particle and provides no indication of the proximity between two different particles, for example one particle attached to one biomolecule and the other particle attached to another biomolecule. Hence these references describe particles that include FRET dye pairs but do not comprise FRET particle pairs.
While such methods have achieved certain degrees of success in their particular applications, there remains a need for a method in which separate, different brightly fluorescent nanoparticles that comprise FRET particle pairs are brought together in close proximity by targeting biomolecules and detected.